Photoimmunotheranostic agents for triple-negative breast cancer diagnosis and therapy that can be activated on demand

Abstract

Triple-negative breast cancer (TNBC) is a heterogeneous disease in which the tumors do not express estrogen receptor (ER), progesterone receptor (PgR) or human epidermal growth factor receptor 2 (HER2). Classical receptor-targeted therapies such as tamoxifen or trastuzumab are therefore unsuitable and combinations of surgery, chemotherapy and/or radiotherapy are required. Photoimmunotheranostics is a minimally invasive approach in which antibodies deliver nontoxic photosensitizers that emit light to facilitate diagnosis and produce cytotoxic reactive oxygen species to induce apoptosis and/or necrosis in cancer cells. We developed a panel of photoimmunotheranostic agents against three TNBC-associated cell surface antigens. Antibodies against epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM) and chondroitin sulfate proteoglycan 4 (CSPG4) were conjugated to the highly potent near-infrared imaging agent/photosensitizer IRDye®700DX phthalocyanine using SNAP-tag technology achieving clear imaging in both breast cancer cell lines and human biopsies and highly potent phototherapeutic activity with IC50values of 62-165 nM against five different cell lines expressing different levels of EGFR, EpCAM and CSPG4. A combination of all three reagents increased the therapeutic activity against TNBC cells by up to 40%.

Keywords: antibody-based therapy; breast cancer; molecular targeting; photodynamic therapy; theranostics.

Figure 1. Fusion proteins labeling with BG-PEG₂₄-IR700

(A) SDS-PAGE followed by fluorescence visualization and (B) subsequent Coomassie staining of BG-PEG₂₄-IR700 or BG-Vista Green coupled to the SNAP-tag fusion proteins. Lanes 1–3: Fusion proteins (scFv-425-SNAP-tag, anti-EpCAM(scFv)-SNAP-tag and anti-CSPG4(scFv)-SNAP-tag) were labeled with a two-fold molar excess of BG-PEG₂₄-IR700. Lanes 4–6: The same proteins were labeled with BG-Vista Green. Lanes 7–9: The SNAP-tag blocking reagent BTP was added to the fusion proteins, followed by incubation with a two-fold molar excess of BG-PEG₂₄-IR700, then a two-fold molar excess of BG-Vista Green. M: Prestained Protein Marker Broad Range (7−175 kDa). Fluorescence visualization of Vista Green (green bands) and IR700 (red bands) was achieved using the CRi Maestro multispectral imaging system.

Figure 2. Stability of fluorescence-labeled scFv-425-SNAP protein in mouse serum

(A) The IR700 fluorescence signal was detected using the CRi Maestro multispectral imaging system (black arrow). M: Prestained Protein Marker Broad Range (7−175 kDa). Lanes 1–8: scFv-425-SNAP-IR700 incubated with mouse serum for 0, 0.5, 1, 2, 3, 4, 5 and 6 h. Lane 9: scFv-425-SNAP-IR700 positive control. (B) Corresponding SDS-PAGE gel showing scFv-425-SNAP-IR700 stained with Coomassie Brilliant Blue.

Figure 3. Analysis of EGFR, EpCAM and CSPG4 protein expression in MDA-MB-468, MDA-MB-231, MDA-MB-453, HS578T and MCF7 cell lines

Flow cytometry was carried out after incubating the cells with Alexa Fluor^®647-labeled scFv-SNAP fusion proteins against EGFR (black curves), EpCAM (dotted black curves) and CSPG4 (gray curves) for 30 min at 37°C in PBS. The filled gray curves represent the non-stained control.

Figure 4. Confocal microscopy showing fluorescence-labeled scFv-SNAP fusion proteins targeting EGFR, EpCAM and CSPG4

The cells were incubated with 0.5 μg of each labeled scFv-SNAP-tag fusion protein (red signal) for 60 min at 37°C and then with Hoechst 33342 nuclear counterstain (blue signal).

Figure 5. Fluorescence immunohistochemistry of scFv-SNAP-Alexa Fluor^®647-labeled proteins against EGFR, EpCAM and CSPG4 in human breast cancer tissues (A) and normal breast tissue (B)

Biopsies were incubated with scFv-425-SNAP-Alexa Fluor^®647, anti-EpCam(scFv)-SNAP-Alexa Fluor^®647 or anti-CSPG4(scFv)-SNAP-Alexa Fluor^®647 overnight at 4°C. A TCS SP5 confocal microscope was used to visualize Alexa Fluor^®647 signals (red) on the breast cancer tissues.

Figure 6. Toxic activity of photoimmunotheranostic agents against four TNBC cell lines and MCF-7

(A) The cytotoxicity of scFv-425-SNAP-IR700 (▼), anti-EpCAM(scFv)-SNAP-IR700 (■), anti-CSPG4(scFv)-SNAP-IR700 (●), the mixture of all three photoimmunotheranostic agents (□), IR700 only (Δ) and the mixture of all three photoimmunotheranostic reagents applied in the absence of NIR irradiation (▲) was evaluated by cell viability assays using four different TNBC cell lines (MDA-MB-468, MDA-MB-453, MDA-MB-231 and Hs758T) and one ER⁺ cell line (MCF-7). Cells were incubated with increasing concentrations of each reagent, separately or in combination, or with IR700 (0, 10, 25, 50, 100, 200 and 400 nM). (B) Induction of apoptosis by photoimmunotheranostic agents (separately) and by IR700, assessed using the Apo-ONE^TM Homogeneous Caspase-3/7 assay.